title: Anti-HBV treatment induces novel reverse transcriptase mutations with reflective effect on HBV S antigen.
abstract: BACKGROUND
  The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification.
  
  
  METHODS
  356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay.
  
  
  RESULTS
  Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:-9.63 kcal/mol, rtA181T:-9.30 kcal/mol, rtA181V:-7.96 kcal/mol, rtS78T:-7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate.
  
  
  CONCLUSIONS
  Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures.
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", 00100 Rome, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Cento
authors.forename: Formijn
authors.initials: F
authors.lastname: Van Hemert
authors.forename: Maria
authors.initials: M
authors.lastname: Neumann-Fraune
authors.forename: Carmen
authors.initials: C
authors.lastname: Mirabelli
authors.forename: Velia-Chiara
authors.initials: VC
authors.lastname: Di Maio
authors.forename: Romina
authors.initials: R
authors.lastname: Salpini
authors.forename: Ada
authors.initials: A
authors.lastname: Bertoli
authors.forename: Valeria
authors.initials: V
authors.lastname: Micheli
authors.forename: Guido
authors.initials: G
authors.lastname: Gubertini
authors.forename: Sara
authors.initials: S
authors.lastname: Romano
authors.forename: Michela
authors.initials: M
authors.lastname: Visca
authors.forename: Giuseppe-Maria
authors.initials: GM
authors.lastname: De Sanctis
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Marino
authors.forename: Francesco
authors.initials: F
authors.lastname: Mazzotta
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Cappiello
authors.forename: Alberto
authors.initials: A
authors.lastname: Spanò
authors.forename: Cesare
authors.initials: C
authors.lastname: Sarrecchia
authors.forename: Francesca
authors.initials: F
authors.lastname: Ceccherini-Silberstein
authors.forename: Massimo
authors.initials: M
authors.lastname: Andreoni
authors.forename: Mario
authors.initials: M
authors.lastname: Angelico
authors.forename: Jens
authors.initials: J
authors.lastname: Verheyen
authors.forename: Carlo Federico
authors.initials: CF
authors.lastname: Perno
authors.forename: Valentina
authors.initials: V
authors.lastname: Svicher
mesh_terms: D000225:Adenine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D000328:Adult; D002460:Cell Line; D018389:Codon, Nonsense; D024882:Drug Resistance, Viral; D005260:Female; D015870:Gene Expression; D006509:Hepatitis B / Q000188:drug therapy*; D006514:Hepatitis B Surface Antigens / Q000032:analysis* / Q000235:genetics; D006515:Hepatitis B virus / Q000187:drug effects*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D063065:Organophosphonates / Q000008:administration & dosage / Q000494:pharmacology; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000008:administration & dosage* / Q000378:metabolism / Q000494:pharmacology; D012641:Selection, Genetic; D017211:Treatment Failure
chemical_list: D018389:Codon, Nonsense; D006514:Hepatitis B Surface Antigens; D063065:Organophosphonates; D018894:Reverse Transcriptase Inhibitors; C053001:adefovir; D012194:RNA-Directed DNA Polymerase; D000225:Adenine
keywords: HBV drug-resistance; HBsAg secretion; HBsAg stop codon; HBsAg structure; Treatment failure